Laura Ezquerra

Yohimbine prevents morphine-induced changes of glial fibrillary acidic protein in brainstem and α2-adrenoceptor gene expression in hippocampus

The alpha(2)-adrenoceptor antagonist yohimbine is known to oppose to several pharmacological effects of opioid drugs, but the consequences and the mechanisms involved remain to be clearly established. In the present study we have checked the effects of yohimbine on morphine-induced alterations of the expression of key proteins (glial fibrillary acidic protein, GFAP) and genes (alpha(2)-adrenoceptors) in rat brain areas known to be relevant in opioid dependence, addiction and individual vulnerability to drug abuse. Rats were treated with morphine in the presence or absence of yohimbine. The effects of the treatments on GFAP expression were studied by immunohistochemical staining in Locus Coeruleus (LC) and Nucleus of the Solitary Tract (NST), two important noradrenergic nuclei. In addition, drug effects on alpha(2)-adrenoceptor gene expression were determined by real time RT-PCR in the hippocampus, a brain area that receives noradrenergic input from the brainstem. Morphine administration increased GFAP expression both in LC and NST as it was previously reported in other brain areas. Yohimbine was found to efficiently prevent morphine-induced GFAP upregulation. Chronic (but not acute) morphine downregulated mRNA levels of alpha(2A)- and alpha(2C)-adrenoceptors in the hippocampus, while simultaneously increased the expression of the alpha(2B)-adrenoceptor gene. Again, yohimbine was able to prevent morphine-induced changes in the levels of expression of the three alpha(2)-adrenoceptor genes. These results correlate the well-established reduction of opioid dependence and addiction by yohimbine and suggest that this drug could interfere with the neural plasticity induced by chronic morphine in central noradrenergic pathways.

The neurotrophic factor pleiotrophin modulates amphetamine-seeking behaviour and amphetamine-induced neurotoxic effects: evidence from pleiotrophin knockout mice

Pleiotrophin (PTN), a neurotrophic factor with important roles in survival and differentiation of dopaminergic neurons, is up‐regulated in the nucleus accumbens after amphetamine administration suggesting that PTN could modulate amphetamine‐induced pharmacological or neuroadaptative effects. To test this hypothesis, we have studied the effects of amphetamine administration in PTN genetically deficient (PTN −/−) and wild type (WT, +/+) mice. In conditioning studies, we found that amphetamine induces conditioned place preference in both PTN −/− and WT (+/+) mice. When these mice were re‐evaluated after a 5‐day period without amphetamine administration, we found that WT (+/+) mice did not exhibit amphetamine‐seeking behaviour, whereas, PTN −/− mice still showed a robust drug‐seeking behaviour. In immunohystochemistry studies, we found that amphetamine (10 mg/kg, four times, every 2 hours) causes a significant increase of glial fibrillary acidic protein positive cells in the striatum of amphetamine‐treated PTN −/− mice compared with WT mice 4 days after last administration of the drug, suggesting an enhanced amphetamine‐induced astrocytosis in the absence of endogenous PTN. Interestingly, we found in concomitant in vitro studies that PTN (3 µM) limits amphetamine (1 mM)‐induced loss of viability of PC12 cell cultures, effect that could be related to the ability of PTN to induce the phosphorylation of Akt and ERK1/2. To test this possibility, we used specific Akt and ERK1/2 inhibitors uncovering for the first time that PTN‐induced protective effects against amphetamine‐induced toxicity in PC12 cells are mediated by the ERK1/2 signalling pathway. The data suggest an important role of PTN to limit amphetamine‐induced neurotoxic and rewarding effects.

Noradrenergic and opioidergic alterations in neuropathy in different rat strains

The Fischer 344 (F344) rat strain differs from the Lewis strain in the response to neuropathic pain. Recently, we found that F344 rats totally recover from mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve 28 days after surgery whereas Lewis rats are initiating their recovery at this time point. Thus, the use of this neuropathic pain model in these different rat strains constitutes a good strategy to identify possible target genes involved in the development of neuropathic pain. Since differences between Lewis and F344 rats in their response to pain stimuli in acute pain models have been related to differences in the endogenous opioid and noradrenergic systems, we aimed to determine the levels of expression of key genes of both systems in the spinal cord and dorsal root ganglia (DRG) of both strains 28 days after CCI surgery. Real time RT-PCR revealed minimal changes in gene expression in the spinal cord after CCI despite the strain considered, but marked changes in DRG were observed. A significant upregulation of prodynorphin gene expression occurred only in injured DRG of F344 rats, the most resistant strain to neuropathic pain. In addition, we found a significant downregulation of tyrosine hydroxylase and proenkephalin gene expression levels in both strains whereas delta-opioid receptor was found to be significantly downregulated only in injured DRG of Lewis rats although the same trend was observed in F344 rats. The data strongly suggest that dynorphins could be involved in strain differences concerning CCI resistance.

Different pattern of pleiotrophin and midkine expression in neuropathic pain: Correlation between changes in pleiotrophin gene expression and rat strain differences in neuropathic pain

Pleiotrophin (PTN) and midkine (MK) are two growth factors highly redundant in function that exhibit neurotrophic actions and are upregulated at sites of nerve injury, both properties being compatible with a potential involvement in the pathophysiological events that follow nerve damage (i.e. neuropathic pain). We have tested this hypothesis by comparatively studying PTN and MK gene expression in the spinal cord and dorsal root ganglia (DRG) of three rat strains known to differ in their behavioural responses to chronic constriction injury (CCI) of the sciatic nerve: Lewis, Fischer 344 (F344) and Sprague–Dawley (SD). Real time RT-PCR revealed minimal changes in PTN/MK gene expression in the spinal cord after CCI despite the strain considered, but marked changes were detected in DRG. A significant upregulation of PTN gene expression occurred in injured DRG of the F344 strain, the only strain that recovers from CCI-induced mechanical allodynia 28 days after surgery. In contrast, PTN was found to be downregulated in injured DRG of SD rats, the most sensitive strain in behavioural studies. These changes in PTN were not paralleled by concomitant modifications of MK gene expression. The results demonstrate previously unidentified differences between PTN and MK patterns of expression. Furthermore, the data suggest that upregulation of PTN, but not MK, could play an important role in the recovery from CCI.

Targeting the Pleiotrophin/Receptor Protein Tyrosine Phosphatase β /ζ Signaling Pathway to Limit Neurotoxicity Induced by Drug Abuse

This review compiles the scientific basis to propose the pleiotrophin/receptor protein tyrosine phosphatase β/ζ signaling pathway as a new therapeutic target to prevent drug of abuse-induced toxicity. In addition, potential guidelines are provided for the development of new therapeutic compounds derived from that knowledge. This approach may be relevant since efficient therapeutic strategies are currently lacking in this field, even when drug-induced neurotoxicity seems to underlie the neurodegenerative disorders diagnosed in drug addicts.

Changes in BDNF gene expression correlate with rat strain differences in neuropathic pain

The Fischer 344 (F344) rat inbred strain differs from the inbred Lewis and the outbred Sprague-Dawley (SD) in the response to different pain stimuli, which has been partially attributed to differences in the endogenous opioid and noradrenergic systems. Since brain-derived neutrophic factor (BDNF) modulates both the endogenous opioid and noradrenergic systems, we have now studied specific changes in BDNF gene expression related to the maintenance of neuropathic pain in the three rat strains. F344 rats were found to be the only strain that completely recovered from neuropathic pain (mechanical allodynia) 28 days after chronic constriction injury (CCI) of the sciatic nerve. Real time RT-PCR studies revealed minimal changes in the expression of BDNF in the spinal cord after CCI despite the strain considered, but marked changes in dorsal root ganglia (DRG) were observed. A significant upregulation of BDNF gene expression was found only in injured DRG of F344 rats, thus correlating with higher resistance to neuropathic pain. The data suggest that BDNF could be involved in strain differences concerning CCI resistance.

Uncovering new pharmacological targets to treat neuropathic pain by understanding how the organism reacts to nerve injury.

The neuropathic pain syndrome is complex. Current drugs to treat neuropathic pain, including anticonvulsivants and antidepressants, fail in up to 40-50% of the patients, while in the rest of them total alleviation is not normally achieved. Increased research advances in the neurobiology of neuropathic pain have not translated in more successful pharmacological treatments by the moment, but recent progress in the experimental methods available for this purpose could result in significant advances in the short term. One rational possibility for the pharmaceutical development of new drugs, including target identification, drug design and evaluation studies, could be to focus on mimicking what organism does to limit nerve damage or to enhance the regeneration of injured axons. Following this strategy, neurotrophic factors such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have been postulated as potential pharmacological targets to treat neuropathic pain. In addition, during the last few years, strong scientific evidences point to novel neurotrophic factors, such as pleiotrophin (PTN), as important factors to limit neuropathic pain development because of their remodeling and angiogenic actions in the injured area. This review focuses on recent research advances identifying new pharmacological targets in the treatment of the cause, not only the symptoms, of neuropathic pain.